The impact of genomic distance on enhancer‐promoter interactions at the CFTR locus

Abstract We identified and characterized multiple cell‐type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the −35 kb airway enhancer and the CFTR promoter in the 16HBE14o− airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and −35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o− cells.


Figure S1 :
Figure S1: Sequence alignments of 16HBE14o -deletion clones.Sanger sequence alignments of deletion targeted areas are shown for 16HBE14o --18.5kbD7.1kb clones (A) or -16kb D3.7kb clones (B).Location and sequence of gRNAs is also shown.PCR amplicons were sequenced directly from genomic DNA, or from amplicons that were cloned into pSCA to distinguish multiple alleles.The allele deletion size(s) in each clone are also shown.

Figure S2 :
Figure S2: Extended analysis of interactions of the -18.5kbD7.1kb deletion clones with the 5' TAD boundary in 16HBE14o -cells.4C-seq analysis of 16HBE14o -WT (grey) and -18.5kbD7.1kb clones (brown) with the viewpoint at the -80.1kb 5' TAD boundary (red dotted line).Key CFTR CREs as well as the deletion are shown at the top.Read quantification tracks from an average of two replicates are shown for each cell type (grey or single colored tracks) and a representative domainogram immediately below.Interaction profile subtraction tracks, for each deletion clone with respect to WT 16HBE14o -are shown in log2 scale.Losses (above) and gains (below) in interactions from 16HBE14o -WT are shown with respect to the y-axis.Regions of interest are marked by horizontal bars or arrows.

Figure S5 :
Figure S5: Extended analysis of the interactions of the -16kb D3.7kb deletion clones with the -20.9kb insulator in 16HBE14o -cells.4C-seq analysis of 16HBE14o -WT (grey) or -16kb D3.7kb clones (teal) with the viewpoint at the -20.9kb insulator (red dotted line).See Figure S2 legend for detailed description of tracks shown.

Figure S6 :
Figure S6: Extended analysis of the interactions of the -18.5kbD7.1kb deletion clones with the CFTR promoter in 16HBE14o -cells.4C-seq analysis of 16HBE14o -WT (grey) and -18.5kbD7.1kb clones (brown) with the viewpoint at the CFTR promoter (red dotted line).See Figure S2 legend for detailed description of tracks shown.

Figure S7 :
Figure S7: Extended analysis of the interactions of the -16kb D3.7kb deletion clones with the CFTR promoter in 16HBE14o -cells.4C-seq analysis of 16HBE14o -WT (grey) and -16kb D3.7kb clones (teal) with the viewpoint at the CFTR promoter (red dotted line).See Figure S2 legend for detailed description of tracks shown.

Figure S8 :
Figure S8: Sequence alignments of Caco2 deletion clones.(A-B) Sanger sequence alignments of deletion targeted areas are shown for Caco2 185+2.7kbD5.1kb clones (A) or 185+5.7kbD2.1kb clones (B).Location and sequence of gRNAs is also shown.Sequencing products were generated from PCR amplicons of genomic DNA, or from amplicons that were cloned into pSCA to distinguish multiple alleles.Deletion size of each clone or identified allele is also shown.Note that 185+5.7kbD2.1kb clone 78 has complex alleleswith one allele having a secondary deletion upstream of the targeted deletion (allele 1) and another allele having a complex inverted sequence inserted upstream of the 5' cut site (blue letters), that contains an independent internal deletion (allele 4).

Figure S10 :
Figure S10: Impact of the 185+2.7kbD5.1kb and 185+5.7kbD2.1kb deletions on interactions with the CFTR promoter in Caco2 cells.4C-seq analysis of Caco2 WT (grey), 185+2.7kbD5.1kb clones (purple), or 185+5.7kbD2.1kb clones (gold) with the viewpoint at the CFTR promoter (red dotted line).Key CFTR CREs as well as the deletions are shown at the top and bottom.Read quantification tracks from an average of two replicates is shown for each cell type (grey or single colored tracks) along with a representative domainogram immediately below.Subtraction tracks, in log2 scale, are shown for each deletion clone interaction profile from Caco2 WT cells.Losses (above) and gains (below) in interactions from Caco2 WT are shown with respect to the y-axis.

Figure S11 :
Figure S11: Extended analysis of interactions of the 185+2.7kbD5.1kb deletion clones with the 5' TAD boundary in Caco2 cells.4C-seq analysis of Caco2 WT (grey) and 185+2.7kbD5.1kb clones (purple) with the viewpoint at the -20.9kb insulator (red dotted line).Key CFTR CREs as well as the deletion are shown at the top.Read quantification tracks from an average of two replicates is shown for each cell type (grey and single colored tracks) along with a representative domainogram immediately below.Subtraction tracks, in log2 scale, are shown for each deletion clone interaction profile from Caco2 WT cells.Losses (above) and gains (below) in interactions from Caco2 WT are shown with respect to the y-axis.Regions of interest marked by horizontal bars or arrows.